Use of nitroaniline derivatives for the production of nitric oxide

ABSTRACT

The present invention relates to the use of a nitroaniline derivative of Formula I for the production of nitric oxide and for the preparation of a medicament for the treatment of a disease wherein the administration of nitric oxide is beneficial. The present invention furthermore relates to a method for the production of NO irradiating a nitroaniline derivative of Formula I, a kit comprising a nitroaniline derivative of Formula I and a carrier and to a system comprising a source of radiations and a container associated to a nitroaniline derivative of Formula I. In Formula I, R and R I  are each independently hydrogen or a C 1 -C 3  alkyl group; R II  is hydrogen or an alkyl group.

BACKGROUND

1. Technical Field

The present disclosure relates to the use of a nitroaniline derivativefor the production of nitric oxide, in particular in the preparation ofa medicament for treating diseases wherein the administration of nitricoxide may be beneficial. The present disclosure further relates to a kitcomprising a nitroaniline derivative and to a system for the productionof nitric oxide.

2. Description of the Related Art

Nitric oxide (NO) has been shown to play a significant role in theregulation of many biochemical pathways in organisms (“Nitric oxide:Physiology, Pathophysiology, and Pharmacology”, M. Moncada et al,Pharmacological Review, vol. 43(2), pages 109-142, 1991 and“Biosynthesis and Metabolism of Endothelium-derived Nitric Oxide”, L. J.Ignarro, Ann. Rev. Pharmacol. Toxicol., vol. 30, pages 535-560, 1990).Nitric oxide has been proven to be an important modulator of vascular,cardiovascular, nervous and immune systems as well as other homeostaticmechanisms (“Inhaled Nitric Oxide: A selective pulmonary vasodilatorreversing human hypoxic K pulmonary vasoconstriction (HPV)”, Z.Blomquist, Circulation, vol. 84, No. 4, page 361, 1991 and “Involvementof nitric acid in the reflex relaxation of the stomach to accommodatefood or fluid”, K. M. Desai et al., Nature, vol. 351, No. 6, page 477,1991). Desai et al demonstrated that adaptive relaxation in isolatedstomach of the guinea pig is mediated by a non-adrenergic,non-cholinergic (NANC) neurotransmitter. Furthermore, they showed thatthis NANC neurotransmitter is undistinguishable from nitric oxidederived from L-arginine. The authors concluded that it is likely thatnitric oxide is a final common mediator of smooth muscle relaxation.

Smooth muscles are present, for example, in the walls of the bloodvessels, bronchi, gastrointestinal tract, and urogenital tract.Administration of nitric oxide gas to the lung by inhalation couldproduce localized smooth muscle relaxation without systemic sideeffects. This characteristic can be used in medicine to treat bronchialconstriction and pulmonary hypertension, pneumonia, etc.

To date, conventional treatment of pulmonary and cardiovascularabnormalities has primarily involved the use of bronchodilators drugs.

Bronchodilators, such as beta-agonists and anticholinergic drugs, areused to reduce airway reactivity and to reverse bronchospasm caused by avariety of diseases, such as asthma, exacerbations of chronic pulmonaryobstructive disease, allergic and anaphylactic reactions and others.

Beta-agonists induce bronchodilation by stimulating receptors thatincrease adenyl cyclase concentrations and the production ofintracellular cyclic adenosine monophosphate (AMP). They can bedelivered by aerosol, orally or parenterally. Administration of theseagents causes significant adverse cardiac effects such as tachycardia,heart palpitations, changes in blood pressure and also other sideeffects including anxiety, tremors, nausea and headaches. Newerbeta2-selective agonists have fewer side effects and somewhat sloweronset of action.

Anticholinergic drug, administered by aerosol, are effectivebronchodilators with relatively few side effects. However, they have aslow onset of action, and 60 to 90 minutes may be required before peakbronchodilation is achieved.

Nitric oxide is unique in that it combines a rapid onset of actionoccurring within seconds with the absence of systemic effects. Onceinhaled, it diffuses through the pulmonary vasculature into thebloodstream, where it is rapidly inactivated by combination withhemoglobin. Therefore, the bronchodilator effects of inhaled nitricoxide are limited to the airway and the vasodilatory effects of inhalednitric oxide are limited to the pulmonary vasculature.

The usage of inhaled NO gas as a selective therapeutic agent for thetreatment of pulmonary and cardiovascular ailments is also reported in“Inhaled nitric oxide as a cause of selective pulmonary vasoldilation inpulmonary hypertension”, J. Perke-Zaba et al, The Lancet, vol. 338, No.9, page 1173, 1991. It has recently been established that theadministration of 5 to 80 ppm of NO in respiratory gases drasticallyimproves persistent pulmonary hypertension of newborn children within afew minutes. This important medical application of NO gas is discussedin “Inhaled nitric oxide in persistent pulmonary hypertension of thenewborn” by J. D. Roberts et al., The Lancet, vol. 340, pages 818-819,1992.

In addition to these effects, NO has also been reported to act asefficient anticancer agent that inhibits key metabolic pathways to blockthe growth of or to kill cells (C. M. Maragos, J. M. Wang, J. A. Hrabie,J. J. Oppenheim, L. K. Keefer, Cancer Res. 1993, vol. 53, page 564; J.B. Mitchell, D. A. Wink, W. DeGraff, J. Gamson, L. K. Keefer, M. C.Krishna, Cancer Res. 1993, vol. 53, page 5845; L. Li, R. G. Kilbourn, J.Adams, I. J. Fidler, Cancer Res. 1991, vol. 52, page 2531; R. J.Griffin, C. W. Song, Presented at the 43rd Annual Meeting of theRadiation Research Society, San Jose, Calif., April 1995; AbstractP15-204; D. Moncada, D. Lekieffre, B. Arvin, B. Meldrum, Neuroreports1993, vol. 343, page 530; D A Wink, Y Vodovotz, J Laval, F Laval, M WDewhirst, and JB Mitchell, Carcinogenesis vol. 19, no. 5, page 711-721,1998).

The failure of NO therapy to achieve widespread usage is primarilyattributable to the previous lack of a precision NO gas generatorsuitable for clinical and biomedical applications.

It is known to produce NO through different methods such as thermalmethods, electrochemical methods or photochemical methods.

Molecular systems useful in thermal methods are extensively studied inthe literature (Peng George Wang, Ming Xian, Xiaoping Tang, Xuejun Wu,Zhong Wen, Tingwei Cai, and Adam J. Janczuk, Chem. Rev. 2002, vol. 102,pages 1091-134; Zhelyaskov, V. R., Godwin, D. W., and Gee, K.,Photochem. Photobiol., 1998, vol. 67, pages 282-288). The maindisadvantage of these methods is that the NO release is not controllableand can not be achieved on demand.

Therefore, the implementation of these methods in medical device is notsuitable and to date it does not exist in any medical device based onsuch methods.

Electrochemical and electrical methods allow accurate delivery ofvariable concentrations of NO upon electrical stimuli. They can allow acontrollable on demand release of NO.

However, the implementation of these methods in devices for medicalapplications gives rise to complex systems, often not applicable toclinical or home use and in some cases based on process susceptible tofluctuations in internal and external operating parameters.

In this context, historically NO gas has been commercially manufacturedusing the well-known Ostwald process in which ammonia is catalyticallyconverted to NO and nitrous oxide at a temperature above 800 DEG C. TheOstwald process is discussed in U.S. Pat. Nos. 4,272,336; 4,774,069 and5,478,549. The Ostwald process, while suitable for the mass productionof NO at high temperatures in an industrial setting, is clearly notapplicable to clinical or home use. Other methods of NO gas generationare based on Haber-Bosch synthesis, as described in U.S. Pat. No.4,427,504, or by taking advantage of paramagnetic properties of nitrousoxide, as described in U.S. Pat. No. 4,139,595.

None of these techniques is suitable for clinical or home use andsignificant industrial application thereof has not been reported. Yetanother method for the generation of NO, which has found limited use inanalytical laboratories, relies upon the reaction of 8 molar nitric acidwith elemental copper. This method is described by F. A. Cotton in thetext “Advanced Inorganic Chemistry”, 5th edition, pages 321-323, JohnWiley & Sons, New York, 1988.

There have been recent attempts to devise apparatus for accuratelydelivering variable concentrations of NO.

By way of example, U.S. Pat. No. 5,396,882 describes a proposal for thegeneration of NO in an electric discharge in air. In the implementationof this proposed technique, electrodes would be separated by an air gapin an arc chamber. The establishment of a high voltage across the airgap would produce a localized plasma for breaking down oxygen andnitrogen molecules and thereby generate a mixture of NO, ozone and otherNO_(x) species. In theory, the concentration of NO could be varied byadjusting the operating current. The gas mixture produced by the processwould be purified and mixed with air in order to obtain therapeuticallysignificant concentrations of NO for administration to a patient. Theprocess proposed in U.S. Pat. No. 5,396,882 would, however, inherentlybe susceptible to fluctuations in internal and external operatingparameters, particularly the ambient humidity. Since the therapeuticallyuseful range of NO concentration is relatively small, it is imperativethat the concentration of administered NO be precisely controlled. Inthe process of U.S. Pat. No. 5,396,882, for example, the achievement ofsuch control would dictate that the NO concentration be closelymonitored at all times. Since the weight of NO generated by the processof U.S. Pat. No. 5,396,882 will vary with fluctuations in operatingparameters, the monitoring of NO concentration would, at best, beextremely difficult and expensive to achieve. Indeed, achemiluminescence analyzer would have to be incorporated into theapparatus and the size and cost of such an analyzer would adverselyaffect the cost and portability of the apparatus.

U.S. Pat. No. 5,827,420 describes an electrochemical process based onthe production of NO through the coulometric reduction of copper (II)ions (Cu²) in a solution of nitric acid accompanied by purging thereaction chamber with an inert gas such as nitrogen. The method permitsprecise control over the rate of production of nitric oxide and can beused for free-standing, portable coulometric generator of controllableamounts of high purity nitric oxide. Nevertheless, such an apparatus isa complex system (several reaction cells, cylinder for carrier gas) thatneeds the presence of several toxic solutions at high concentration (8Mnitric acid, 0.1 CuSO₄).

Photochemical methods allow the release of NO in a controllable andprecise way on demand upon light stimuli. There are a limited number ofcompounds able to generate NO using light as trigger that are known inthe state of the art (Bordini J., Hughes D. L., Da Motta Neto J. D.,Jorge da Cunha C., Inorg. Chem., 2002, vol. 41(21), pages 5410-5416; G.Stochel, A. Wanat, E. Kulis, Z. Stasicka, Coord. Chem. Rev., 1998, vol.171, pages 203-220; S. Wecksler, A. Mikhailovsky, P. C. Ford, J. Am.Chem. Soc., 2004, vol. 126, pages 13566-13567; J. Baurassa, W. DeGraff,S. Kudo, D. A. Wink, J. B. Mitchell, P. C. Ford, J. Am. Chem. Soc. 1997,vol. 119, page 2853; K. M. Miranda, X. Bu, I. Lorkovic, P. Ford, Inorg.Chem. 1997, vol. 36, page 4838; V. R. Zhelyaskov, K. R. Gee, D. W.Godwin, Photochem. Photobiol. 1998, vol. 67, page 282; L. R. Makings, R.Y. Tsien, J. Biol. Chem. 1994, vol. 269, page 6282; D. J. Sexton, A.Muruganandam, D. J. McKenney, B. Mutus, Photochem. Photobiol., 1994,vol. 59, page 463; M. C. Frost, M. E. Meyerhoff, J. Am. Chem. Soc. 2004,vol. 126, pages 1348-1349; T. Suzuki, O, Nagae, Y. Kato, H. Nakagawa, K.Fukuhara, N. Miyata, J. Am. Chem. Soc., 2005, vol. 127, pages11720-11726).

Among these, the majority is activated by light in the ultra-violet(UV)-range that, is not only biologically dangerous, but also requirescomplex instrumentation and is not suitable to be integrated inminiaturized and portable devices.

Only limited examples of NO donors activated by visible light are known(Valentin R. Zhelyaskov and Dwayne W. Godwin, “NITRIC OXIDE: Biology andChemistry”, Vol. 2, No. 6, pages 454-459 (1998); Bordini J., Hughes D.L., Da Motta Neto J. D., Jorge da Cunha C., Inorg. Chem., 2002, vol.41(21), pages 5410-5416; J. Baurassa, W. DeGraff, S. Kudo, D. A. Wink,J. B. Mitchell, P. C. Ford, J. Am. Chem. Soc. 1997, vol. 119, page2853). However, their chemical structures are not adequate for easychemical derivatization in order to allow their assembly onto films andnanoparticles. Moreover, one potential disadvantage of these compoundsis related to the toxic effect of their photoproducts. This requirescomplex apparatus in order to avoid diffusion of the toxic cagingmoiety.

Finally, it is known to use a compound of Formula

commonly known as Flutamide, for the production of NO via UV-visiblelight irradiation (S. Sortino, S. Petralia, G. Compagnini, S. Conoci andG. Condorelli, Light-Controlled Nitric Oxide Generation from a NovelSelf-Assembled Monolayer on Gold Surface, Angew. Chem. Int. Ed.2002-41/11, 1914-1917).

Disadvantageously, this compound shows some limitations related to thefact that only a very small portion of its absorption falls in thevisible region of the electromagnetic spectrum and therefore longirradiation times and expensive devices are needed to obtain acceptableyields of NO.

BRIEF SUMMARY

Certain embodiments provide the use of new compounds able to generate NOand a method for the production of NO that are free from the abovedescribed drawbacks and in particular are free from toxic effects andallow for an easy derivatization.

One embodiment provides a method comprising:

converting nitroaniline derivatives of Formula (I) to nitric oxide:

wherein, R and R^(I) are each independently hydrogen or a C₁-C₃ alkylgroup; and R^(II) is hydrogen or a substituted or unsubstituted alkylgroup, the converting including irradiating the nitroaniline derivativesof Formula (I) with radiation having a wavelength between 300 nm to 500nm.

Another embodiment provides a method of administering nitric oxide gasto a subject in need thereof, comprising:

generating nitric oxide gas by irradiating a nitroaniline derivative ofFormula (I) with radiation having a wavelength of about 300 nm to 500nm:

wherein, R and R^(I) are each independently hydrogen or a C₁-C₃ alkylgroup; and R^(II) is hydrogen or a substituted or unsubstituted alkylgroup; and

introducing the nitric oxide gas into an airway of the subject.

A further embodiment provides compounds well-suited to be employed inminiaturized, low cost and portable medical devices.

One embodiment provides a kit comprising:

a nitroaniline derivative of Formula (I):

wherein, R and R^(I) are each independently hydrogen or a C₁-C₃ alkylgroup; R^(II) is hydrogen or a substituted or unsubstituted alkyl group;and

a carrier.

Another embodiment provides a system comprising:

a source of radiation having a wavelength of between 300 and 500 nm;

a support or container associated to a nitroaniline derivative ofFormula (I):

wherein R and R^(I) are each independently hydrogen or a C₁-C₃ alkylgroup; R^(II) is hydrogen or a substituted or unsubstituted alkyl group;and

a carrier.

A further embodiment provides a method comprising:

administering nitric oxide gas to a subject in need thereof, theadministering including: generating nitric oxide gas by irradiating anitroaniline derivative of Formula (I) with radiation having awavelength of about 300 nm to 500 nm:

wherein R and R^(I) are each independently hydrogen or a C₁-C₃ alkylgroup; and R^(II) is hydrogen or a substituted or unsubstituted alkylgroup; and introducing the nitric oxide gas into an airway of thesubject.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 shows the absorption spectrum of the nitroaniline derivatives ofFormula (I).

FIG. 2 shows the concentration of NO released from an aqueous solutionof NOME 10⁻⁴M detected with a fluorimetric as a function of time.

FIG. 3 shows the absorption spectra of4-nitro-3-(trifluoromethyl)aniline in methanol measured every 30 minutesover a two-hour period.

FIG. 4 shows the fluorescent spectrum that indicates the presence ofnitrites and therefore the formation of NO following irradiation of a4-nitro-3-(trifluoromethyl)aniline solution.

FIG. 5 shows absorbance, measured at 30 minute intervals, of a1043-(trifluoromethyl)-4-nitrophenylamino)decan-1-thiol in a CH₃OH:H₂O(10%) solution.

FIG. 6 shows the fluorescent spectrum that indicates the presence ofnitrites and therefore the formation of NO following irradiation of a10-(3-(trifluoromethyl)-4-nitrophenylamino)decan-1-thiol solution.

FIG. 7 shows the absorption spectrum of photoactive Pt-nanoparticles.

FIG. 8 shows the NO photo-release profile of nanoparticlesfunctionalized with1043-(trifluoromethyl)-4-Nitrophenylamino)decan-1-thiol.

FIG. 9 shows an apparatus for the delivery of the nitroanilinederivatives of Formula (I) in aerosol form.

DETAILED DESCRIPTION

Certain embodiments describe a nitroaniline derivative of Formula (I), amethod for the production of nitric oxide using the same, a kit and asystem including the same, and a method of administering nitric oxide.

In particular, one embodiment provides a method of converting anitroaniline derivative of Formula (I) to nitric oxide, the convertingcomprising: irradiating the nitroaniline derivatives of Formula (I) withradiation having a wavelength between 300 nm to 500 nm:

wherein:

R and R^(I) are each independently hydrogen or a C₁-C₃ alkyl group;

R^(II) is hydrogen or a substituted or unsubstituted alkyl group.

In certain embodiments, R^(II) is Ak-Y, wherein Ak is a branched orunbranched C₂-C₁₈ alkyl group optionally substituted with —OH or NH₂,and Y is a tail-group selected from the group consisting of hydrogen,halogen, —SH, —S—SR^(III), —Si(OR^(IV) ₃)₃, —Si—X₃, and—CH═CR^(V)R^(VI), wherein R^(III) is selected from the group consistingof alkyl group, aryl group, aralkyl group, alkenyl group, alkynyl groupand heterocyclic group; R^(N) is an alkyl group; R^(V) and R^(VI) areindependently hydrogen or alkyl group; X is halogen, more preferablychlorine or bromine. Preferably, R^(III) is an alkyl group, morepreferably a C₁-C₃₀ alkyl group.

As used herein, “alkyl” or “Ak” refers to a straight (i.e., unbranched)or branched hydrocarbon chain radical comprising carbon and hydrogenatoms, containing no unsaturation, having from one to thirty carbonatoms (i.e., C₁-C₃₀ alkyl). In certain embodiments, an alkyl maycomprise two to eighteen carbon atoms (i.e., C₂-C₁₈ alkyl). In otherembodiments, an alkyl may comprise one to three carbon atoms (i.e.,C₁-C₃ alkyl). The alkyl is attached to the rest of the molecule by asingle bond, for example, methyl (Me), ethyl (Et), n-propyl,1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl(t-butyl), 3-methylhexyl, 2-methylhexyl, and the like.

“Alkenyl” refers to a straight or branched hydrocarbon chain radicalgroup comprising carbon and hydrogen atoms, containing at least onedouble bond, and having from two to twelve carbon atoms. In certainembodiments, an alkenyl may comprise two to eight carbon atoms. In otherembodiments, an alkenyl may comprise two to four carbon atoms. Thealkenyl is to the rest of the molecule by a single bond, for example,ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl,pent-1-enyl, penta-1,4-dienyl, and the like.

“Alkynyl” refers to a straight or branched hydrocarbon chain radicalgroup comprising carbon and hydrogen atoms, containing at least onetriple bond, having from two to twelve carbon atoms. In certainembodiments, an alkynyl may comprise two to eight carbon atoms. In otherembodiments, an alkynyl has two to four carbon atoms. The alkynyl isattached to the rest of the molecule by a single bond, for example,ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.

“Aryl” refers to a radical derived from an aromatic monocyclic ormulticyclic hydrocarbon ring system by removing a hydrogen atom from aring carbon atom. The aromatic monocyclic or multicyclic hydrocarbonring system contains only hydrogen and carbon from six to eighteencarbon atoms, where at least one of the rings in the ring system isfully unsaturated, i.e., it contains a cyclic, delocalized(4n+2)π-electron system in accordance with the Hückel theory. Arylgroups include, but are not limited to, groups such as phenyl,fluorenyl, and naphthyl.

“Aralkyl” refers to an alkyl (as defined herein) substituted with anaryl group, for example, benzyl, diphenylmethyl and the like.

“Halogen” refers to bromo, chloro, fluoro or iodo.

“Heterocyclic group” refers to a stable 3- to 18-membered non-aromaticring radical that comprises two to twelve carbon atoms and from one tosix heteroatoms selected from nitrogen, oxygen and sulfur. Unless statedotherwise specifically in the specification, the heterocyclyl radicalmay be a monocyclic, bicyclic, tricyclic or tetracyclic ring system,which may include fused or bridged ring systems. The heteroatoms in theheterocyclyl radical may be optionally oxidized. One or more nitrogenatoms, if present, may be optionally quaternized. The heterocyclylradical may be fully saturated (e.g., tetrahydrofuranyl), partiallyunsaturated or fully unsaturated (e.g., pyridinyl). The heterocyclyl maybe attached to the rest of the molecule through any atom of the ring(s).

“Optionally substituted” means that, any of the above-described radicalgroup may or may not be substituted with one or more substituents.Unless specified otherwise, the description of any of the above radicalgroups (e.g., alkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclicgroup) includes both unsubstituted radicals and radicals substitutedwith one or more of the following groups: halogen, cyano, nitro, oxo,thioxo, trimethylsilanyl, —OR^(a), —Si(OR^(a))₃, —SR^(a), —OC(O)—R^(a),—N(R^(a))₂, —C(O)R^(a), —C(O)OR^(a), —C(O)N(R^(a))₂,—N(R^(a))C(O)OR^(a), —N(R^(a))C(O)R^(a), —N(R^(a))S(O)_(t)R^(a) (where tis 1 or 2), —S(O)_(t)OR^(a) (where t is 1 or 2) and —S(O)_(t)N(R^(a))₂(where t is 1 or 2) where each R^(a) is independently hydrogen, alkyl,aryl, aralkyl, heterocyclic group.

According to one embodiment, the nitroaniline derivative of Formula (I)is 4-nitro-3-(trifluoromethyl)aniline.

The method allows to produce in very precise and controlled way nitricoxide.

Advantageously, nitroaniline derivatives of Formula (I) present anexcellent solubility in water solution and a very high chemicalstability over a wide range of pH (tested via HPLC-ESI-MS). Moreover,they allow an easy derivatization. All these properties are essentialprerequisites for biological applications in medical devices, inparticular for the employment in miniaturized low cost and portabledevices.

Further, the photochemical nitroaniline derivatives of Formula (I) areable to produce NO by visible light irradiation, in particular whenirradiated with radiations having a wavelength of between 300 and 500nm, preferably between 400 and 450 nm.

Advantageously, nitroaniline derivatives of Formula (I) present a verygood overlap of their absorption spectrum with the visible region ofelectromagnetic spectrum (see FIG. 1) in contrast to the know compoundsand, in particular, the aforementioned Flutamide.

The shift of the absorbance curve of nitroaniline derivatives of Formula(I) compared to the absorbance curve of Flutamide is caused by thesubstitution of the amide group of Flutamide with an amine group.

This substitution was proved to cause a great variation of the molarextinction coefficient of the chromophore and the shift of theabsorbance towards longer wavelengths, thus allowing the moleculeexcitation with light having lower frequency.

Advantageously, the inventors found that the substitution of the amidegroup of Flutamide with an amine group does not affect the ability ofthe photochemical group of nitroaniline derivatives of Formula (I) toproduce NO when irradiated with appropriated wavelengths.

In particular, they have found that nitroaniline derivatives of Formula(I), when irradiated with radiations having a wavelength of between 300and 500 nm, undergo a radical reaction to generate NO, as shown in thescheme below.

The mechanism of NO photo-generation of nitroaniline derivatives ofFormula (I) is based on a nitro-to-nitrite photo-rearrangement followedby the formation of a phenoxy radical as the main transient intermediateand a phenol derivative as a stable end product in agreement withliterature.

At the end of the reaction, 4-hydroxy-3-(trifluoromethyl)anilinederivatives are produced. These photoproducts are phenol-derivativesthat, according to similar compounds, are potential antioxidants. Thisovercomes the toxicity problems of photoproducts of other NOphoto-generators (Bordini J., Hughes D. L., Da Motta Neto J. D., Jorgeda Cunha C., Inorg. Chem., 2002, vol. 41(21), pages 5410-5416; G.Stochel, A. Wanat, E. Kulis, Z. Stasicka, Coord. Chem. Rev., 1998, vol.171, pages 203-220; K. M. Miranda, X. Bu, I. Lorkovic, P. Ford, Inorg.Chem. 1997, vol. 36, page 4838).

In particular, the ability of nitroaniline derivatives of Formula (I) togenerate NO upon 400 nm light irradiation is reported in FIG. 2.

FIG. 2 represents the concentration of NO released, detected with afluorimetric assay (T. P. Misko, R. J. Schilling, D. Salvemini, W. M.Moore, M. G. Currie, Anal. Chem., 1993, 214, 11.), as a function of timefrom an aqueous solution of NOME 10⁻⁴M.

Because their absorption spectra are in the visible range of the light,nitroaniline derivatives of Formula (I) can be integrated in a verysmall, portable device.

Moreover, the use of visible light instead of UV, permits directapplication in biological sample without any risk of phototoxicity.

The nitroaniline derivatives of Formula (I) can be in a liquid or in asolid form and can be associated to a carrier. The carrier preferablyhas a minor molar absorptivity in the wavelength of between 300 and 500nm, i.e., less than 15%, preferably 10%, more preferably less than 5% ofthat of the nitroaniline derivative associated therewith.

The carrier can be a liquid solution, for example an aqueous solution,or a solid substrate, for example in the form of nanoparticles orbi-dimensional surfaces, selected from the group consisting of a metal,an inorganic oxide, silicon and a plastic polymeric material.

The preferred metals are selected from the group consisting of gold,platinum and silver, and the preferred inorganic oxides are siliconoxide (SiO_(x)) or indium tin oxide (ITO).

In one embodiment, the nitroaniline derivatives of Formula (I) arechemically bound to the solid substrate, provided that the formation ofan amide bond between the amine group of the nitroaniline derivative ofFormula (I) and the solid substrate is avoided. In fact, the presence ofa carboxy group adjacent to the amine group of the nitroanilinederivatives of Formula (I) has proved to cause the shift of theabsorbance of the nitroaniline derivatives of Formula (I) to wavelengthsin the UV spectrum.

Preferably, when the tail-group of nitroaniline derivatives of Formula(I) is —SH or —S—SR^(II), a metallic substrate can be used as a carrier;when the tail-group is —Si(OR^(III) ₃)₃ or —Si—X₃, inorganic oxides canbe used as a solid substrate and when the tail-group is—CH═CR^(Iv)R^(V), a silicon substrate can be used.

The solid substrate can be, for example, in the form of nanoparticlesbearing the nitroaniline derivatives of Formula (I) on their surface, inthe form of bi-dimensional elements on which a layer of a nitroanilinederivative of Formula (I) is deposited or in the form of a porous solid,soaked with a nitroaniline derivative of Formula (I).

The present disclosure also relates to the use of a nitroanilinederivative of Formula (I) as described above, for the preparation andadministration of NO for the treatment of a disease wherein theadministration of nitric oxide is beneficial.

Preferably the disease can be selected in the group consisting ofrespiratory diseases, cancers, and vascular diseases.

As described above, nitroaniline derivatives of Formula (I) can beassociated to a carrier, preferably having minor molar absorptivity inthe wavelength range of between 300 and 500 nm.

For example, it is possible to use as a carrier an aqueous solution ofmetal nanoparticles covalently coated with nitroaniline derivatives ofFormula (I) that can be firstly delivered in the human body and thenexternally (to the human body) irradiated. This is particularly suitablefor in vivo application.

One embodiment is a method for the production of nitric oxide comprisingthe step of irradiating a nitroaniline derivative of Formula (I), asdescribed above, with radiations having a wavelength comprised between300 and 500 nm, preferably between 400 and 450. The nitroaniline ofFormula (I) can be associated to one of the above-mentioned carriers,transparent to visible light.

One embodiment is a kit comprising a nitroaniline derivative of Formula(I) and a carrier as described above.

One embodiment is a system comprising a source of radiations having awavelength of between 300 and 500 nm and a support or containerassociated to the above-described nitroaniline derivative of Formula(I).

Advantageously, while molecular systems that use UV light need verylarge and expensive lamps, also having a short lifetime, such asfluorescent lamps, nitroaniline derivatives of Formula (I) can work witha source of radiations such as a blue light emitting diode (LED) that isvery small (about 2 mm or smaller), low cost (2-4 dollars) and has avery long lifetime (up to 100K hours).

This permits the use of nitroaniline derivatives of Formula (I) in aportable device for in situ application.

The support or container can be, for example, a chamber made ofoptically transparent material containing an aqueous solution ofnitroaniline derivatives of Formula (I) at appropriate concentration (upto mM range) or a two dimensional solid substrate bearing a film ofnitroaniline derivatives of Formula (I) either in a monolayer form(obtained by self assembling) or in a thicker form (obtained by spincoating) or even a porous solid soaked with nitroaniline derivatives ofFormula (I) in a liquid form.

The solid substrate can be, for example, an inorganic base material suchas glass, silicon, or quartz, or a plastic polymeric material.

A scheme of one of the possible systems in which nitroanilinederivatives of Formula (I) can work is shown in FIG. 9.

In detail, FIG. 9 shows an apparatus 1 for the delivery of thenitroaniline derivatives of Formula (I) in aerosol form. The apparatus 1comprises a container 2 for an aqueous solution 3 of nanoparticlesbearing the nitroaniline compound. A source of visible light, e.g., anarray 4 of blue LEDs, is arranged below the container 2 and is connectedto a power supply (not shown). An inhalator 5 is arranged above thecontainer 2 for administering the nitric oxide generated by theirradiation of the aqueous solution 3 to a patient.

Further characteristics of the present disclosure will appear in thefollowing description of merely illustrative and not limiting examples.

Example 1 NO Photo-Release of 4-Nitro-3-(trifluoromethyl)aniline

4-nitro-3-(trifluoromethyl)aniline was dissolved in methanol and thesolution was irradiated in a quartz cuvette with 8 lamps emittingradiations with wavelength of 350 nm for 2 hours. The absorbance of thesolution was measured every 30 minutes.

It was observed a progressive reduction of the absorbance peak in theband having the lowest energy associated to an increase of theabsorbance peak in the band having the highest energy (FIG. 3)

In order to demonstrate the NO release in solution, the Damiani method(P. Damiani, G. Burini, Talanta 1986, vol. 33, page 649) convenientlymodified by Misko (T. Misko, R. J. Schilling, D. Salvemini, W. Moore, M.Currie, Anal. Biochem. 1993, vol. 214, page 11) was used. It allows theindirect determination of NO by measuring nitrite levels. In particular,NO is an highly reactive gas that, in the presence of water, forms anequimolar mixture of nitrites and nitrates. Nitrites, reacting with aDAN (2,3-diaminonaphtalene) solution in the presence of an acid, form1-naphthotriazole, a highly fluorescent compound having a characteristicUV absorbance.

Therefore, the solution of 4-nitro-3-(trifluoromethyl)aniline inmethanol after irradiation was dried and then the recovered4-nitro-3-(trifluoromethyl)aniline was re-dissolved with water andphosphate buffer 0.1M (pH 7.4). 150 μl of a DAN solution were added tothe solution of 4-nitro-3-(trifluoromethyl)aniline heating at 50° C. for40 minutes. Subsequently, 150 μl of concentrated NaOH was added in orderto obtain naphthotriazole ions according to the scheme below.

The fluorescent spectrum of the solution thus obtained revealed thepresence of nitrites and therefore the formation of NO after irradiationof 4-nitro-3-(trifluoromethyl)aniline (FIG. 4).

As shown in FIG. 4, emission peaks of 1-naphtotriazole were clearlyidentified.

Example 2 NO Photo-Releasing of10-(3-(trifluoromethyl)-4-Nitrophenylamino)decan-1-thiol

10-(3-(trifluoromethyl)-4-Nitrophenylamino)decan-1-thiol was dissolvedin a CH₃OH:H₂O (10%) solution. This solution was irradiated in a quartzcuvette with 8 lamps emitting radiations with wavelength of 350 nm for35 minutes. The absorbance of the solution was measured every 30 minutes(FIG. 5).

300 μl of a DAN saturated solution in HCl 0.6 M were added to 3 ml ofthe irradiated solution of10-(3-(trifluoromethyl)-4-Nitrophenylamino)decan-1-thiol followed byheating at 50° C. for 20 minutes.

Subsequently, 1 ml of NaOH was added to 1 ml of the solution in order toobtain naphthotriazole ions. The fluorescent spectrum of the solutionthus obtained revealed the presence of nitrites and therefore theformation of NO after irradiation of10-(3-(trifluoromethyl)-4-Nitrophenylamino)decan-1-thiol (FIG. 6). Asshown in FIG. 6, emission peaks of 1-naphtotriazole were clearlyidentified.

Example 3 Preparation of Pt-Nanoparticles Functionalized with10-(3-(Trifluoromethyl)-4-Nitrophenylamino)decan-1-thiol

Pt-nanoparticles were prepared according to the scheme below.

These nanoparticles are soluble in water. TEM analysis revealed thepresence of particles having a diameter of about 1 nm.

The functionalization of Pt-nanoparticles as described above with10-(3-(trifluoromethyl)-4-Nitrophenylamino)decan-1-thiol may be carriedout according to the scheme below.

FIG. 7 reports the absorption spectrum of the photoactivePt-nanoparticles.

The spectrum clearly shows the typical plasmon absorption band of Ptalong with the typical absorption of the photoactive unit at about 420nm. This proves that the above schemed synthesis was effective.

The NO photo-release of the nanoparticles functionalized with1043-(trifluoromethyl)-4-Nitrophenylamino)decan-1-thiol was measuredusing fluorimetric assay based on 2,3-diaminonaphthalene (DAN) [T. P.Misko, R. J. Schilling, D. Salvemini, W. M. Moore, M. G. Currie, Anal.Chem., 1993, vol. 11, pages 214], as shown in Examples 1 and 2 (FIG. 8).

The curve shows the same trend (linear behavior) of the aqueous solutionof 10-(3-(trifluoromethyl)-4-Nitrophenylamino)decan-1-thiol.

The Pt-nanoparticles can be used as a delivery agent or carrier for invivo applications.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A kit comprising a nitroaniline derivative of Formula (I):

R and R^(I) are each independently hydrogen or a C₁-C₃ alkyl group;R^(II) is hydrogen or a substituted or unsubstituted alkyl group; and acarrier.
 2. The kit according to claim 1, wherein: R^(II) is Ak-Y,wherein Ak is a branched or unbranched C₂-C₁₈ alkyl group optionallysubstituted with —OH or NH₂, and Y is a tail-group selected from thegroup consisting of hydrogen, halogen, —SH, —S—SR^(III), —Si(OR^(IV)₃)₃, —Si—X₃, and —CH═CR^(V)R^(VI); R^(III) is selected from the groupconsisting of alkyl group, aryl group, aralkyl group, alkenyl group,alkynyl group and heterocyclic group; R^(III) is an alkyl group; R^(V)and R^(VI) are independently hydrogen or alkyl group; and X is halogen.3. The kit according to claim 1, wherein said nitroaniline derivative ofFormula (I) is 4-nitro-3-(trifluoromethyl)aniline.
 4. The kit accordingto claim 1, wherein said carrier has a minor molar absorptivity in thewavelength comprised between 300 and 500 nm.
 5. The kit according toclaim 1, wherein said carrier is a liquid solution.
 6. The kit accordingto claim 1, wherein said carrier is a solid substrate selected from thegroup consisting of a metal; an inorganic oxide; and a plastic polymericmaterial.
 7. The kit according to claim 6, wherein said nitroanilinederivative of Formula (I) is chemically bound to said solid substratewithout forming an amide bond.
 8. The kit according to claim 7, whereinwhen said tail-group is —SH or —S—SR^(III), K said solid substrate is ametal.
 9. The kit according to claim 7, wherein when said tail-group is—Si(OR^(III) ₃)₃, or —Si—X₃, said solid substrate is an inorganic oxide.10. The kit according to claim 7, wherein when said tail-group is—CH═CR^(V)R^(VI), said solid substrate is silicon.
 11. A systemcomprising: a source of radiation having a wavelength of between 300 and500 nm; a support or container associated to a nitroaniline derivativeof Formula (I):

wherein R and R^(I) are each independently hydrogen or a C₁-C₃ alkylgroup; R^(II) is hydrogen or a substituted or unsubstituted alkyl group;and a carrier.
 12. The system according to claim 11, wherein: R^(II) isAk-Y, wherein Ak is a branched or unbranched C₂-C₁₈ alkyl groupoptionally substituted with —OH or NH₂, and Y is a tail-group selectedfrom the group consisting of hydrogen, halogen, —SH, —S—SR^(III),—Si(OR^(IV) ₃)₃, —Si—X₃, and —CH═CR^(V)R^(VI); R^(III) is selected fromthe group consisting of alkyl group, aryl group, aralkyl group, alkenylgroup, alkynyl group and heterocyclic group; R^(IV) is an alkyl group;R^(V) and R^(VI) are independently hydrogen or alkyl group; and X ishalogen.
 13. The system according to claim 11, wherein said nitroanilinederivative of Formula (I) is 4-nitro-3-(trifluoromethyl)aniline.
 14. Thesystem according to claim 11, wherein the source of radiation comprisingone or more light emitting diodes.
 15. A system comprising: a source ofradiation having a wavelength of between 300 and 500 nm; a support orcontainer associated to a nitroaniline derivative of Formula (I):

wherein R and R^(I) are each independently hydrogen or a C₁-C₃ alkylgroup; R^(II) is hydrogen or a substituted or unsubstituted alkyl group;and a solid substrate, wherein the nitroaniline derivative of Formula(I) is chemically bound to the solid substrate.
 16. The system accordingto claim 15 wherein R^(II) is Ak-Y, wherein Ak is a branched orunbranched C₂-C₁₈ alkyl group optionally substituted with —OH or NH₂,and Y is a tail-group selected from the group consisting of hydrogen,halogen, —SH, —S—SR^(III), —Si(OR^(IV) ₃)₃, —Si—X₃, and—CH═CR^(V)R^(VI); R^(III) is selected from the group consisting of alkylgroup, aryl group, aralkyl group, alkenyl group, alkynyl group andheterocyclic group; R^(IV) is an alkyl group; R^(V) and R^(VI) areindependently hydrogen or alkyl group; and X is halogen.
 17. The systemaccording to claim 16, wherein said nitroaniline derivative of Formula(I) is 4-nitro-3-(trifluoromethyl)aniline.
 18. The system according toclaim 15, wherein the source of radiation comprising one or more lightemitting diodes.
 19. The system according to claim 15 wherein the solidsubstrate is a metal, a semiconductive material or an inorganic oxide.